Meropenem in combination with baicalein exhibits synergism against extensively drug resistant and pan-drug-resistant Acinetobacter baumannii clinical isolates in vitro

Abstract Several studies have demonstrated that the effectiveness of carbapenems against drug-resistant Acinetobacter baumannii infections has been decreasing. Combination therapy with two or more drugs is currently under investigation to overcome the emerging resistance against carbapenems. In this study, we tested the possible synergistic interactions of a potent antibacterial flavonoid, baicalein, with meropenem to illustrate this duo’s antibacterial and antibiofilm effects on 15 extensively drug resistant or pan-drug-resistant (XDR/PDR) A. baumannii clinical isolates in vitro. Isolates included in the study were identified with MALDI-TOF MS, and antibiotic resistance patterns were studied according to EUCAST protocols. Carbapenem resistance was confirmed with the modified Hodge test, and resistance genes were also analyzed with genotypical methods. Then, checkerboard and time-kill assays were performed to analyze antibacterial synergism. Additionally, a biofilm inhibition assay was performed for screening the antibiofilm activity. To provide structural and mechanistic insights into baicalein action, protein–ligand docking, and interaction profiling calculations were conducted. Our study shed light on the remarkable potential of the baicalein–meropenem combination, since either synergistic or additive antibacterial activity was observed against every XDR/PDR A. baumannii strain in question. Furthermore, the baicalein–meropenem combination displayed significantly better antibiofilm activity in contrast to standalone use. In silico studies predicted that these positive effects arose from inhibition by baicalein of A. baumannii beta-lactamases and/or penicillin-binding proteins. Overall, our findings highlight the prospective potential benefits of baicalein in combination with meropenem for the treatment of carbapenem-resistant A. baumannii infections.


Introduction
With its exceptional capacity to cause chronic and nosocomial infections Acinetobacter baumannii is a bacterium posing se v er e threat to public health. Even last-resort antibiotics and their combinations are becoming ineffective with an increasing pace (Assimakopoulos et al. 2019 ), placing this pathogen among the most criticall y r esistant bacteria according to the World Health Organization (WHO 2017 ) and stressing the immediacy of the need for ne w tr eatment str ategies.
Combined use of carbapenems or fluoroquinolones has gained popularity instead of using the antibiotic alone especially against m ultidrug-r esistant (MDR), extensiv el y drug r esistant (XDR; MDR plus resistance to carbapenems), and pan-drug-resistant (PDR; XDR plus resistance to polymyxins) A. baumannii strains (Kar aisk os and Giamar ellou 2014 , Kar ak onstantis et al. 2021 , Huang et al. 2022 ). Furthermor e, antimicr obial monother a py is not suggested in infections caused by colistin-resistant A. baumannii strains in clinical practice (Hong et al. 2016 ). At this point, alternativ e nov el str ategies wher e antibiotics ar e combined with antimicr obiall y efficient pol yphenols , peptides , and phages ha ve emerged and gained momentum over the years against the abovementioned resistant A. baumannii strains (Jubair et al. 2021, Cara wa y et al. 2022, Meng et al. 2022, Sisakhtpour et al. 2022.
Baicalein (BCL) (5,6,7-trihydr oxy-2-phen ylc hr omen-4-one) is a trihydroxyflavone obtained from the root extracts of Scutellaria baicalensis . Pr e vious inv estigations hav e pr oposed that BCL possess antimicrobial activity in addition to many other health benefits such as antio xidant, anti-inflammatory, antihypertensi ve, and anticancer properties (Wan et al. 2018 ). Furthermore , prior studies ha ve demonstrated the great potential of BCL as a potent antimicrobial, and it has significant effects on disruption of biofilm structur e, inactiv ation of virulence factors such as quorum sensing, bacteriocins etc. against various pathogens (Cai et al. 2016, Luo et al. 2016b, Vijayakumar et al. 2021. In search of a novel strategy, we propose that BCL may have an improved antimicrobial action when combined with an antibiotic that is curr entl y a viable ther a py option. This strategy should pr ov e useful in (i) tr eating antibiotic-r esistant infections, (ii) limiting the emergence of antibiotic resistance, and (iii) managing the side effects of antibiotics, such as toxicity, due to reduced c hemother a peutic concentr ations.
In this study, a series of clinical XDR and PDR A. baumannii isolates was first subjected to detailed microbiological characterization with the aim of profiling their antibiotic resistance phenotypicall y and genotypicall y. Next, the combined antimicr obial and antibiofilm activity of meropenem (MEM) and BCL against these isolates was assessed by using in vitro experiments. Last, in an in silico setting, the ability of BCL to bind A. baumannii OXAs (oxacillinase type of beta-lactamases) and PBPs (Penicillin binding protein) was predicted based on molecular docking calculations.

Materials
Mueller-Hinton broth (MHB) (cat. no: 1.10293.0500), blood agar base (cat. no: 1.10886.0500), and Mueller-Hinton agar (MHA) (cat no: 1.05437.0500) were purchased from Merck (Germany) and were used for cultivating the bacteria and/or for the assa ys . BCL (P.N:465119) and MEM (P.N: 32 460) were purchased from Sigma-Aldrich (USA) and stored according to the manufacturer's recommendations. BCL was dissolved in dimethyl sulfoxide (final concentration did not exceed 5% in all experiments), and MEM was dissolved in sterile distilled water. Glycerol supplemented brain heart infusion broth (cat. no: M210-500G) was purc hased fr om Himedia (India) and used for stor a ge of bacterial isolates. BD-Crystal ® Enteric/Non-fermenter Identification kit was used for confirmation of pure isolation of isolates. Escherichia coli ATCC 25922 was used as the bacterial strain sensitive to imipenem in the modified Hodge test (MHT). All solutions' pH values were c hec ked and adjusted r outinel y after pr epar ation of all solutions and before usage in the assa ys .

Bacterial isolates and characterization
Bacterial strains used in this study were isolated from Çukurova University Hospital in Turkey. Acinetobacter baumannii strains were identified at the species level by matrix-assisted laser desor ption/ionization-time-of-flight mass spectr ometry (MALDI-TOF MS), and initial antibiotic resistance patterns were determined by the MicroScan WalkAway plus System (Beckman Coulter, USA).

Antibiotic resistance profiling and confirmation of carbapenem resistance
The antibiotic resistance profiles of the selected strains were established by measuring the limit values for various antibiotics with the Kirby-Bauer disk diffusion method according to the EU-C AST criteria (EUC AST 2015 ). Resistance c har acteristics of the str ains wer e e v aluated accor ding to the EUCAST guidelines b y measuring zone diameters after 18-20 h of incubation (EUCAST 2015 ).
Str ains that wer e found to be r esistant to carba penems wer e further e v aluated with MHT to confirm carba penem r esistance as described pr e viousl y (Girlic h et al. 2012 ).

Genotypic analysis of antibiotic resistance
Nucleic acid extraction of the strains was ac hie v ed by boiling and a ppl ying the fr eeze-thawing method without using an y c hemicals (Chen et al. 2020 ). Then pol ymer ase c hain r eaction (PCR) targeting the bla OXA-51 gene was performed to confirm the identification of the isolates as carbapenemase producer A. baumannii . Isolates w ere sear ched for antibiotic resistance genes using multiplex PCR panels to detect Ambler class bla genes and mobile genetic elements. Primers were selected to amplify the following bla gene groups: class A, bla TEM , bla SHV , bla CTX-M , bla VEB , bla PER , bla KPC , and bla GES ; class B, bla IMP , bla VIM , bla GIM , bla SPM , bla SIM , and bla NDM ; and class D, bla OXA-23-like , bla OXA-24-like , and bla OXA-51-like as described pr e viousl y (Dallenne et al. 2010 ). For screening colistin resistance, the mcr-1 gene was screened by PCR method by using primers as described pr e viousl y . All PCR assays were carried out by using Thermo Scientific Taq green master mix in Applied Biosystems 2720 thermocycler (Thermo Fisher Scientific, CA, USA). Positive and negative controls were included in all PCR assa ys . Resistance genes pre-detected b y PCR w ere further examined b y DN A sequence analysis, and the specific sequences for the resistance gene were identified based on closest Blast matches from the NCBI database ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ). Sequencing reactions were carried out by using the same primer sets and BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) in an automated Sanger sequencer (ABI 310 Genetic analyzer; Applied Biosystems, Foster City, CA, USA).

Determination of MICs
Br oth micr o dilution assay was conducted to determine the minimum inhibitory concentrations (MICs) of MEM and BCL against the A. baumannii isolates. Briefly, MEM and BCL stock solutions wer e pr epar ed and adjusted to 1 mg/ml. Suspensions of bacterial samples were prepared to 0.5 McFarland turbidity standard and then diluted 100-fold using saline to adjust the final bacterial cell numbers to 1 × 10 6 CFU/ml. A volume of 50 μl of MHB was added to each well of a microplate. Next, 50 μl of test solution was added and diluted serially to wells containing MHB, except the controls. Finally, 50 μl of bacterial suspension was added to all wells, except the negative control. The microplates were incubated at 37 • C for 24 h under aer obic conditions. MIC v alues wer e determined as the lo w est concentration of the antimicrobial agent where the growth of bacteria was inhibited. Results were interpreted according to the EUCAST guidelines (EUCAST 2015 ).

Synergy testing: c hec k erboard and time-kill assays
The putative antimicrobial synergy between MEM and BCL was analyzed by performing a checkerboard assay (Sopirala et al. 2010 ), whic h normall y depends on testing the susceptibility of str ains to differ ent combinations of m ultiple a gents at v arying doses . T he starting concentrations of the agents were 4 × MIC and were diluted until 1/32 × MIC for both for MEM and BCL, respectiv el y. The combined effect of the agents was evaluated by calculating the total fractional inhibitory concentration index ( FICI).
Time-kill kinetic assays were performed only for strains, which displayed highest MICs for MEM, lowest MICs for BCL, and were found to be "synergistic" in the checkerboard assay (Barry et al. 1999 ). Briefly, MHB with agents alone and in combinations were inoculated with an aliquot of the strain with a concentration of ∼10 5 CFU/ml. MEM and BCL were evaluated at 0.5 × MIC, alone and/or combined in addition to contr ol, whic h was 0.1% DMSO. Samples were taken at 0, 2, 4, 8, 12, and 24 h follo w ed b y serial dilution in saline and plating on MHA at an amount of 50 μl. Plates were incubated at 37 • C for 24 h, for CFU counting. Reduction in two or more logarithmic units between the combined and the most activ e a gent alone after 24 h was consider ed as syner gistic.

Biofilm inhibition assay
The biofilm inhibitory potentials of MEM and BCL either alone or in combination with each other against A. baumannii were investigated by using MIC and sub-MIC (MIC/2 or MIC/4) concentrations of the agents in a 96-well plate. Control samples, whic h wer e pr epared with no added drug, were also included in the assa ys . Firstly, 96-well plates containing bacterial samples at a final concentration of 1 × 10 6 cfu/ml and varying concentrations of the agents were incubated for 24 h at 37 • C to generate biofilms. After biofilm production, the w ells w ere gently w ashed three times with saline solution to get rid of planktonic cells and to k ee p only the cells that adhered onto the wells' inner surface. To dye the biofilm layers, 125 μl of 1% (w/v) crystal violet solution was added into e v ery well and incubated for 30 min, follo w ed b y cleaning with saline solution. Finall y, pr e viousl y pr epar ed 95% (v/v) ethanol was added to e v ery well and incubated for 30 min to decolorize the bacteria and to stain the biofilms. Lastly, the absorbance values of the solutions were measured at 570 nm on a Varioskan Flash Multi Detection Microplate Reader (Thermo Fisher Scientific, USA). The obtained absorbance data were analyzed by quantitatively estimating the biofilm inhibition, in which the control samples were taken as standards . T he r esults wer e pr esented as % inhibition of biofilm formation.

Protein-ligand docking and interaction profiling
The 3D conformer of BCL (compound ID: 5281605) in SDF format was r etrie v ed fr om the PubChem open chemistry database a vailable at https://pubchem.ncbi.nlm.nih.go v/ (Kim et al. 2021 ). The crystal structures of (i) A. baumannii OXA-23 in complex with MEM (PDB ID: 4JF4), (ii) A. baumannii OXA-51 (K83D/I129L variant) in complex with doripenem (PDB ID: 5L2F), and (iii) A. baumannii PBP1a in complex with imipenem (PDB ID: 3UDX) were downloaded from the RCSB Protein Data Bank available at https: // www.rcsb.org/ (Burley et al. 2021 ). Hydrogen coordinates and appr opriate pr otonation states wer e assigned to PBP1a and the OXAs of interest by using the Protoss hydrogen prediction tool available at https:// proteins.plus/ (Ertl andSchuffenhauer 2009 , Bietz et al. 2014 ). BCL was docked in the presence of structurally relevant water molecules onto the A. baumannii proteins by using the JAMDA molecular docking tool available at https:// proteins.plus/ (Schellhammer and Rarey 2007 , Henzler et al. 2014, Flac hsenber g et al. 2020. Each binding site was defined by the bona fide carbapenem ligand, with a site radius of 6.5 Å . Molecular docking was executed with medium pr ecision. Favor able non-cov alent inter actions between BCL and the A. baumannii proteins were computed by using Discovery Studio Visualizer, v16.1.0 (Dassault Systèmes BIOVIA Corp ., San Diego , CA, USA).

Sta tistical anal ysis
Data wer e statisticall y anal yzed and visualized by using the Gr a phP ad Prism Softwar e, LLC., Version 9.2.0 (Gr a phP ad Softwar e Inc., San Diego, CA, USA). One-w ay ANOVA follo w ed b y Sidak's multiple comparisons test was employed to analyze the reductions in MIC values. χ 2 -test was used to compare biofilm inhibition percentages among different concentrations. P-values of < .05 were regarded significant, and asterisks were used to indicate significant differences between different samples/sample sets.

Antibiotic resistance profiling demonstr a tes XDR and PDR phenotype and genetic background of the resistance
A total of 15 clinical isolates were selected for this study. The isolates were collected from internal medicine service and cultivated fr om differ ent clinical specimens including aspir ate ( n = 8), blood ( n = 5), wound ( n = 1), and sputum ( n = 1). Isolates were collected from nine males and six females in an a ge r ange between 41 and 76 with a mean age of 66.47 ± 2.34 years. Clinical data of the isolates, their genes responsible for antibiotic resistance, and their antimicr obial r esponses to differ ent antibiotics ar e shown in Table 1 . With respect to carbapenem resistance, all isolates were discov er ed to be resistant to both imipenem and MEM in disk diffusion assa ys . T he results of the MHT further confirmed the carba penem r esistance for the entir e set of isolates.

Combination of MEM and BCL demonstrates synergism or additi v e antibacterial interactions against all isolates
The MIC values of A. baumannii isolates against MEM and BCL were inv estigated by br oth micr odilution assay. As depicted in Table 2 , the results of the assays r e v ealed that A. baumannii isolates had a range of MIC values between 15.60-125 mg/l and 7.80-31.25 mg/l against MEM and BCL, respectively.
The combined inhibitory actions of MEM and BCL on A. baumannii isolates were evaluated by a checkerboard assay firstly. This assay demonstrated that MEM and BCL had synergistic/ad diti ve effects on all isolates. Indifference or antagonism was not observ ed for an y isolate, suggesting a pr oductiv e alliance between the two a gents. Syner gistic inter actions wer e observ ed for 9 out of 15 A. baumannii isolates (46.66%), and ad diti v e inter actions were detected for 6 out of 15 A. baumannii isolates (53.4%). Synergism was applicable to 5 out of 8 PDR A. baumannii isolates, while 4 out of 7 XDR A. baumannii isolates were affected by synergism between the agents. Detailed MICs of agents alone, MICs of agents in combination and FICI values are given in Table 2 and Fig. 1 .
To confirm synergism between MEM and BCL a time-kill assay was performed for thr ee r epr esentativ e str ains namel y, AB1, AB3, and AB7. These three isolates are selected because of being (i) PDR, (ii) highl y r esistant to MEM, (iii) susceptible to BCL, and (iv) found to be synergistic in checkerboard assay. Synergism was observed in three tested strains confirming the results of checkerboard assay. Detailed results of time-kill assay are given in Fig. 1 .

In silico studies shed light to OXA and PBP binding potential of BCL
In an attempt to predict the inhibitory activity of BCL against r esistance-r elated A. baumannii targets, BCL was docked into the active-site clefts of O XA-23, O XA-51, and PBP1a in a "closed" (i.e. carbapenem-bound) conformation. The finest docking solutions were selected according to computed JAMDA scores as well as additional structural criteria already described for bona fide carbapenem ligands (Han et al. 2011, Smith et al. 2013, June et al. 2016. The results of our molecular docking simulations revealed that BCL could be accommodated well in the active-site clefts of OXA-23 (JAMDA score: -2.24887) and OXA-51 (JAMDA score: -2.24221). In the predicted OXA-23-BCL complex (Fig. 3 ), the ligand was demonstrated to form hydrogen-bonding interactions with Thr217, Tr p219, and Ar g259 thr ough its hydr oxyl gr oups (namel y, C5-OH and C6-OH) on the benzene ring (A-ring). Arg259 was able to form an additional hydrogen bond with the carbonyl oxygen on the heterocyclic pyran ring (C-ring) of BCL. Phe110 was found to be engaged in aromatic stacking interactions with the C-ring as well as the phenyl substitution at position 2 (B-ring). A similar interaction pattern was also observed for the predicted OXA-51-BCL complex (Fig. 4 ). Gly219 was found to form a carbon-hydrogen bond with the C5-OH group of the ligand. Trp220 (which corresponds to Trp219 in OXA-23) was able to form a hydrogen bond with the C6-OH group and an ar omatic stac king inter action with the C-ring. The C5-OH group in its deprotonated form (calculated p K a : 8.35) a ppear ed to be salt bridged to Ar g260 (whic h corr esponds to Arg259 in OXA-23). Arg260 could also form a hydro-  gen bond with the carbonyl oxygen on the C-ring. The C and B rings were further stabilized by ar omatic stac king inter actions with Phe111 (whic h corr esponds to Phe110 in OXA-23). Ov er all, these findings support the notion that BCL could possibly exert its synergistic effect by competing with MEM for binding at the active sites of the two most prevalent OXAs in the clinical A. baumannii isolates of question, thereby inhibiting MEM hydrolysis and incr easing the effectiv e concentr ations of MEM in the in vitro setting. An alternative, or most likely an additional, synergistic role play ed b y BCL could be its binding to PBPs in A. baumannii , thus inhibiting cell wall formation and potentiating the antibacterial action of MEM. Our docking calculations r e v ealed that BCL could be housed well in the active-site cleft of PBP1a (JAMDA score: -2.11966) and that it could establish hydrogen-bonding interactions with Ser434, Thr670, and Thr672 through its hydroxyl groups (C5, C6, and C7) on the A-ring (Fig. 5 ). An additional hydrogen bond could be formed between the carbonyl oxygen on the C-ring and Ser470. Tyr707 was found to be an important residue in further stabilizing BCL via aromatic stacking interactions with the A and B rings.

Discussion
In this report, successful sensitization of XDR and PDR A. baumannii isolates by combining MEM with BCL was ac hie v ed, and synergism between the agents was demonstrated in terms of both antibacterial and antibiofilm actions . Furthermore , we provided structural and mechanistic insights into how this synergism might work. The isolates used in this study were eight PDR and se v en XDR A. baumannii str ains, whic h wer e r esistant to all tested antibiotics (except that the XDR strains were only susceptible to colistin) representing major antibiotic classes . T he carbapenem resistance of all isolates was further confirmed by MHT. Genotypical analysis demonstrated a wide arsenal of resistance genes, which were predominantly O XA-51, O XA-23, follo w ed b y O XA-48, O XA-23, TEM, VIM, and IMP, and these r esults wer e corr elated with the susceptibility profiles of the isolates.
Here, we tested the performance of a highly promising compound, BCL in combination with MEM in an effort to overcome antibiotic resistance in a subset of A. baumannii clinical isolates. When combined with BCL, the MIC values of MEM were significantl y r educed. In fact, in vitro test concentr ations ar e v ery instructive for us and can r eadil y be tr anslated to in vivo drug doses, highlighting the additional potential benefits of our combination str ategy, suc h as decreasing the toxicity and/or managing the adverse effects of MEM (Baldwin et al. 2008, Liu et al. 2020. It is worth noting that BCL has almost no toxicity to human cells as reported in earlier studies (Dinda et al. 2017 ). Furthermor e, r eac hing to concentrations as low as 0.97 mg/l for BCL in combination is promising when e v aluated with peak concentr ations ac hie v ed in human serum (Li et al. 2021 ). In our setting, a significant reduction in MEM concentrations was ac hie v ed in killing of the isolates wher e MEM-BCL combination sho w ed synergism or ad diti v e action a gainst all isolates. Mor eov er, the mean MIC v alue of BCL a gainst XDR/PDR isolates was 18.21 mg/l [5.26 mg/l (0.97 mg/l lowest) in combination], whic h was r easonabl y low for a natur al flavonoid. To compar e, in a similar study focusing on carbapenem resistant A. baumannii isolates, quercetin, which is a well-known natural flavonoid for its antimicrobial activity demonstrated MICs around 64 mg/l against tested isolates (Pal and Tripathi 2019 ).
In addition to generating a potent antimicrobial synergism, BCL and MEM are cooperating well against the biofilm structure of the pathogen as well. Biofilm inhibition percentages of the agents wer e significantl y higher in combination compared to standalone a pplications especiall y at lo w er concentr ations. Exopol ysacc haride matrices produced by A. baumannii make the bacteria more resistant to external stressors including antibiotics mainly by making it harder for the antibiotic molecule to r eac h the target cell (Harding et al. 2018 ). In the r ele v ant scientific liter atur e, ther e ar e onl y a fe w studies e v aluating the anti-biofilm activity of BCL against various pathogens . T hese reports suggest that BCL holds the potential to inhibit biofilm formation, destroy biofilms, increase the permeability of various antibiotics, and block quorum sensing in selected pathogens (Chen et al. 2016, Shirley et al. 2017, Vijayakumar et al. 2021. Although most of these studies involv e Gr am-positiv e bacteria or yeasts, our results are consistent with them and considered novel as we provide antibiofilm activity data for BCL against A. baumannii (XDR and PDR) for the first time in this in vestigation. T he mechanism underlying the anti-biofilm property of BCL warrants further tests, but recent studies have suggested different modes of action pertaining to diverse agents in w ell-kno wn pathogens (Chen et al. 2016, Luo et al. 2016a, Najarzadeh et al. 2019. Natur al pr oducts hav e historicall y played a significant r ole in drug discovery. In many instances, BCL was found to restore/enhance the in vitro antibacterial activity of MEM against the A. baumannii clinical isolates in question. The mechanism underl ying this syner gistic effect could be inter pr eted as the putative ability of BCL to inhibit MEM-hydr ol yzing β-lactamases, such as OXAs, in A. baumannii , and/or pr e v ent A. baumannii fr om constructing a cell w all b y binding to peptidoglycan-synthesizing PBPs, in a similar fashion to MEM. Although ad diti v e (and e v en indiffer ent) inter actions ar e also able to minimize antimicr obial r esistance de v elopment in bacteria (Noel et al. 2021 ), the nonspecific mec hanisms underl ying ad diti vity may be difficult to ascertain because of a wide range of cellular targets as well as strain-dependent genetic and biochemical networks.
Using structural information and kinetic data obtained for the OXA-51 I129L variant and other variants from clinical A. baumannii isolates , J une et al. ha v e pr oposed that OXA-51-like enzymes ar e only one to two substitutions a wa y from acquiring kinetic properties similar to or identical to those observed for OXA-23 (June et al. 2016 ). Here, using computational studies, w e sho w that BCL holds the potential to inhibit OXA-23 and OXA-51, two major βlactamases present in the entire set of clinical A. baumannii isolates included in this work. Owing to its trihydr oxyc hr omenone structure and the attached phenyl ring, BCL may also bind to A. baumannii PBP1a and ideally render this cell wall synthesis enzyme inacti ve. Unlik e high-molecular-weight (HMW) class B PBPs that perform only transpeptidase functions, HMW class A PBPs (e.g. PBP1a and PBP1b) are bifunctional enzymes performing both tr anspeptidase and tr ansgl ycosylase functions (Ghuysen 1991 ). Although PBPs pr obabl y make a slight contribution to antibiotic resistance in bacteria, identifying compounds that bind PBPs will e v entuall y pav e the way for the structure-based design of more effective antibiotics. It should be mentioned here, ho w ever, that our in silico findings are yet to be confirmed by further in vitro and/or in vivo studies.
Our r esearc h has se v er al limitations to note. First and foremost, a larger set of isolates including standard strains may be r equir ed to further validate the results. Next, our findings concern the in vitro activity of the investigated antimicrobial combinations and should be understood with the knowledge that in vitro susceptibility data are not the only factor to consider when determining the most optimal antibiotic treatment for patients. Last, because this is a single-center study, it may not accur atel y r eflect the susceptibility of strains with similar c har acteristics identified in elsewhere.
To conclude, our findings provide an important basis for the de v elopment of viable alternative regimens to treat patients suffering from XDR/PDR A. baumannii infections. Future studies are suggested to elucidate other likely interactions and mechanisms behind the combinatory actions of BCL and MEM. Also, further in vivo studies are required to validate the clinical use and effectiveness of this novel treatment strategy.

Ethical statement
This study has the exemption of ethical r e vie w according to the rules of the Health Subcommittee of Eastern Mediterranean Univ ersity Researc h and Publication Ethics Board as there is no involvement of human subjects or human-related biological materials in the study.

Ac kno wledgments
The bacterial isolates used in this study were also studied by the author, T.K., within the specific scope of her doctoral thesis.